Q5® Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Protocol tips
- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Publication protocol

PP1 protein production
Untagged γ‐isoform of the catalytic subunit of human PP1 and its variants were overexpressed and purified using published protocols (Alessi et al, 1993; Barford & Keller, 1994). Transformed E. coli DH5α cells were grown in Luria–Bertani (LB) medium supplemented with 2 mM MnCl2 and 100 μg/ml ampicillin at 30°C until OD600 reached ~0.25. Protein expression was induced with 0.5 mM IPTG. Cells were harvested by centrifugation and resuspended in buffer A (50 mM imidazole, 0.5 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 10% glycerol, 2 mM β‐mercaptoethanol, 2 mM MnCl2, pH 7.5) supplemented with Complete EDTA‐free protease inhibitor cocktail, lysozyme (0.01 mg/ml), and DNase (0.05 mg/ml). After cell lysis, insoluble material was sedimented by centrifugation and the supernatant filtered using 0.22‐μm filter prior to loading on a 5‐ml heparin column equilibrated with buffer A. PP1 was eluted using a 100‐ml gradient to 50% buffer A supplemented with 1 M NaCl. Fractions were analyzed on a 12% SDS–PAGE gel and those containing PP1 were pooled and diluted 10‐fold with buffer C (50 mM imidazole, 0.5 mM EDTA, 0.5 mM EGTA, 10% glycerol, 5 mM β‐mercaptoethanol, 2 mM MnCl2, pH 7.2) for injection in a HiTrapQ HP (GE Healthcare) column. PP1 was eluted using a gradient to 40% buffer C supplemented with 1 M NaCl. PP1 was further purified by size‐exclusion chromatography (SEC) using a Superdex 75 16/60 (GE Healthcare) column equilibrated with SEC buffer (50 mM imidazole, 0.5 mM EDTA, 0.5 mM EGTA, 300 mM NaCl, 10% glycerol, 5 mM β‐mercaptoethanol, 2 mM MnCl2, pH 7.5) for downstream applications. PP1 mutations (PP1 N124D and PP1 D64N) were introduced using the Q5 Site‐Directed Mutagenesis kit (New England Biolabs). All constructs were verified by sequencing. Expression and purification of PP1 variants were carried out as for wild‐type PP1.

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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Site-Directed Mutagenesis Kit below.

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